Nucleic acid formulations for gene delivery and methods of use

ABSTRACT

A nucleic acid formulation for use in gene delivery comprising a nucleic acid and an anionic polymer is disclosed. Examples of the anionic polymer includes anionic amino acid polymer or poly-amino acid (such as poly-L-glutamic acid, poly-D-glutamic acid, poly-L-aspartic acid, poly-D-aspartic acid), poly-acrylic acid, polynucleotides, poly galacturonic acid, and poly vinyl sulfate.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of, and claims priority to, U.S.application Ser. No. 10/234,406 filed Sep. 3, 2002, now U.S. Pat. No.7,173,116 which was the U.S. National Application of and a Continuationof International Patent Application No. PCT/US01/06953, filed Mar. 2,2001 which claims priority to U.S. Provisional Application Ser. No.60/187,236 filed Mar. 3, 2000 and U.S. Provisional Application Ser. No.60/261,751 filed Jan. 16, 2001, which are all hereby incorporated byreference, including any drawings, as if fully set forth herein.

FIELD OF THE INVENTION

This invention relates to novel compositions and methods for theintroduction of a nucleic acid molecule into a cell, including by apulse voltage delivery method, for the expression of a protein, peptide,antisense RNA, ribozyme, or polypeptide.

BACKGROUND OF THE INVENTION

The following information is presented solely to assist theunderstanding of the reader. None of the information is admitted todescribe prior art to the claims of the present invention.

Gene therapy is a major area of research in drug development. Genetherapy has been considered a desirable mechanism to correct geneticallydetermined diseases resulting from the failure to produce certainproteins and acquired diseases such as autoimmunity and cancer. Oneexample of a class of genetically determined diseases that areconsidered amenable to gene therapy is hemophilia. Hemophilia B, forexample, is a bleeding disorder that results from the absence offunctional blood clotting Factor IX (“F.IX”). The disease state isclassified as severe, moderate or mild, depending on the level offunctional F.IX. (Lusher, J. M. (1999) Thromb Haemost 82:572-5751).Approximately 5,200 males are afflicted with the disease in the U.S.with approximately 45% of these cases being of the severe type. Insevere cases of hemophilia B (<1% of normal F.IX levels) there arefrequent bleeding events that can be life threatening and often producedebilitating destruction of the patient's joints. The current therapyfor hemophilia B is the administration of F.IX protein in response tobleeding events only. The use of either blood derived or recombinantF.IX has shown that tremendous clinical and quality of life benefits canbe achieved by converting the most severe hemophilia B cases into themoderate or mild range. In some countries F.IX protein is givenprophylactically in the most severe cases, despite the fact that thesetreatments are extremely expensive (Ljung, R. C. (1999) Thromb Haemost82:525-530). The prophylactic use of F.IX is not frequent in the U.S.

Gene therapy could provide a new prophylactic approach for the treatmentof diseases such as hemophilia B. A technological barrier tocommercialization of gene therapy, however, is the need for practical,effective and safe gene delivery methods. In animal models ofhemophilia, viral-based vectors have been used successfully toadminister the human F.IX gene either to liver or muscle. (Kay, M. A.,et al. (1993) Science 262:117-119; Herzog, R. W., et al. (1999) Nat Med:56-63; Snyder, R. O., et al. (1999) Nat Med 5:64-70; Chao, H., et al.(1999) Gene Ther 6:1695-1704; Lozier, J. N., et al. (1999) Blood94:3968-3975; Kaufman, R. J. (1999) Hum Gene Ther 10:2091-2107). In somecases, these approaches have led to long-term (>2 years) expression oftherapeutic levels of F.IX in a canine model of hemophilia B (Herzog, R.W., et al. (1999) Nat Med 5:56-63). However, the limitations ofviral-based approaches have been extensively reported. For instance,re-administration is not possible with these vectors because of thehumoral immune response generated against the viral proteins. Inaddition to manufacturing challenges to obtain adequate reproduciblevector supply, there are also significant safety concerns associatedwith viral vectors, particularly for those targeting the liver for geneexpression. Not withstanding the problems associated with viral genetherapy, viruses have been considered by many to be more efficient thannon-viral delivery vehicles.

A problem of non-viral gene therapy is to achieve the delivery andexpression of sufficient nucleic acid to result in a tangible,physiologically relevant expression. Although DNA plasmids in isotonicsaline (so-called ‘naked’ DNA) were shown several years ago to transfecta variety of cells in vivo, the lack of stability of such unprotectedplasmids to enzymatic degradation is associated with irreproducibilityin uptake leading to highly variable expression and biological responsesin animal models. The very low bioavailability of ‘naked’ plasmid inmost tissues also requires high doses of plasmids to be administered togenerate a pharmacological response.

The field of non-viral gene delivery has therefore been directed to thedevelopment of more efficient synthetic delivery systems able toincrease the efficiency of plasmid delivery, confer prolonged expressionand provide for storage stable formulations as is expected of otherpharmaceutical formulations.

To overcome the problem of degradation of nucleic acids, typicallyplasmid DNA (“pDNA”), and enhance the efficiency of gene transfection,cationic condensing agents (such as polybrene, dendrimers, chitosan,lipids, and peptides) have been developed to protect pDNA by condensingit through electrostatic interaction. (A. P. Rolland, From genes to genemedicines: recent advances in nonviral gene delivery, review inTherapeutic drug carrier systems, 15(2):143-198 (1998).) However, theuse of condensed plasmid particles for transfection of a large number ofmuscle cells in vivo has not been successful as compared directly to“naked” DNA. Wolff, J. A., et al., J. Cell Sci., 103, 1249, 1992. Inparticular, due to the physiology of the muscle, the use of rigidcondensed particles containing plasmid for efficient transfection of alarger number of muscle cells has not been successful to date becausecationic lipid and polylysine plasmid complexes do not cross theexternal lamina to gain access to the caveolae and T tubules. Id.

Additional strategies that include the modulation of the plasmid surfacecharge and hydrophobicity by interaction with protective, interactivenon-condensing systems (e.g., PINC™ polymers) have shown advantages overthe use of ‘naked’ DNA for direct administration to solid tissues.[WO9621470, U.S. Pat. No. 6,040,295, incorporated herein by reference.]

Biodegradable microspheres have also been used in gene delivery thatencapsulate the nucleic acid. For example, WO0078357, Chen, W. et al,disclosed matrices, films, gels and hydrogels which include hyaluronicacid (HA) derivatized with a dihydrazide and crosslinked to a nucleicacid forming slow release microspheres. WO9524929, Boekelheide, K. etal., disclosed encapsulation of genes in a matrix preferably in the formof a microparticle such as a microsphere, microcapsule, a film, animplant, or a coating on a device such as a stent. U.S. Pat. No.6,048,551, Beer, S. et al. disclosed a controlled release gene deliverysystem utilizing poly (lactide-co-glycolide) (PLGA), hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, and the Ludragit R, L,and E series of polymers and copolymer microspheres to encapsulate thegene vector. Luo D et al. Pharm Res 1999 Aug. 16(8):1300-8, reported thecharacterization of systems for controlled delivery of DNA fromimplantable polymer matrices (EVAc: poly (ethylene-co-vinyl acetate))and injectable microspheres (PLGA and PLA: poly (D,L-lactide-co-glycolide) copolymer and poly (L-lactide), respectively).Despite their promise, microspheres can pose manufacturing difficultiesand can adversely constrain the release of DNA in vivo, particularly inmuscle tissue.

Thus, despite these recent advances, there remains a need for additionaland improved formulated nucleic acid compositions and methods ofadministering the same for gene therapy.

SUMMARY OF THE INVENTION

An alternative approach to the use of viral vectors is the use ofnon-viral plasmid-based gene therapy. The present invention disclosesnovel compositions and methods for enhancing the administration ofnucleic acids and uptake thereof by an organism. In one embodiment, theformulation utilizes anionic polymers such as poly-amino acids,polynucleotides, or poly-acrylic acids that are able to enhance thetransfection of nucleic acids to muscle tissues with and withoutelectroporation. In one embodiment of the invention, the poly-amino acidis poly-glutamic acid and salt thereof. The poly-glutamic acidformulation has been shown in the present invention to be particularlyuseful in increasing electroporation assisted transfection in vivo.

The compositions of the present invention that are used to administernucleic acid, preferably by pulse voltage delivery, allows for treatmentof diseases, vaccination, and treatment of muscle disorders and serumprotein deficiencies.

Another aspect of the present invention provides a method for treating amammalian condition or disease. The method involves the step ofadministering to a mammal suffering from the condition or disease atherapeutically effective amount of a composition of the invention. Inone embodiment of the invention, the disease is characterized byinsufficient levels of active Factor IX. Delivery of a nucleic acidencoding Factor IX formulated in poly-glutamate and delivered inconjunction with electroporation according to the present invention isable to provide nanogram levels of Factor IX in the peripheral blood oflarge animals.

In one embodiment of the invention, the disease is characterized byinsufficient levels of red blood cells resulting in anemia. Delivery ofa nucleic acid encoding erythropoietin (“EPO”) formulated inpoly-L-glutamate and delivered in conjunction with electroporationaccording to the present invention is able to provide sufficient levelsof EPO to result in a maximal hematocrit level.

In one embodiment of the invention, the disease is characterized bydisregulation of the immune system. Delivery of a nucleic acid encodinga cytokine, such as in one example, human interferon alpha 2b (“hINFα”),formulated in poly-L-glutamine and delivered in conjunction withelectroporation according to the present invention is able to providenanogram levels of hINFα in the peripheral circulation.

In yet another aspect, the invention also features a method fordelivering a nucleic acid molecule to a mammal, more preferably a human,by utilizing a non-condensing anionic polyamino acid formulation. Themethod involves the step of providing a composition of the invention tothe cells of the organism by use of a device configured and arranged tocause pulse voltage delivery of the composition.

In preferred embodiments the device for delivering is an electroporationdevice that delivers the composition of the invention to the cell bypulse voltage and/or delivers the composition of the invention bysubjecting the cells to an electric field.

The present invention also features a kit. The kit includes a containerfor providing a composition of the invention and either (i) a pulsevoltage device for delivering the composition of the invention to cellsof an organism, wherein the pulse voltage device is capable of beingcombined with the container, or (ii) instructions explaining how todeliver the composition of the invention with the pulse voltage device.Thus the “container” can include instructions furnished to allow one ofordinary skill in the art to make compositions of the invention. Theinstructions will furnish steps to make the compounds used forformulating nucleic acid molecules. Additionally, the instructions willinclude methods for testing compositions of the invention that entailestablishing if the nucleic acid molecules are damaged upon injectionafter electroporation. The kit may also include notification of an FDAapproved use and instructions.

A method for making a kit of the invention is also provided. The methodinvolves the steps of combining a container for providing a compositionof the invention with either (i) a pulse voltage device for deliveringthe composition of the invention to the cells of an organism, whereinthe pulse voltage device is capable of being combined with thecontainer, or (ii) instructions explaining how to deliver thecomposition of the invention with the pulse voltage device.

The invention also provides a method of treating a mammal suffering fromcancer or an infectious disease. The method involves the step ofproviding a composition of the invention to cells of the mammal by useof a device configured and arranged to provide pulse voltage delivery ofa composition of the invention to cells of the mammal, wherein themolecule encodes a cancer antigen or an antigen for the infectiousdisease.

As noted above, the compositions of the present invention that are usedto administer nucleic acid, preferably by pulse voltage delivery,include a compound that protects the nucleic acid and/or prolongs thelocalized bioavailability of the nucleic acid and/or enhances expressionwhen administered to an organism in vivo, or in vitro in cell culture.

As the compositions are useful for delivery of a nucleic acid moleculeto cells in vivo, in a related aspect the invention provides acomposition at an in vivo site of administration. In particular, thisincludes compositions for delivering a nucleic acid molecule at an invivo site in a mammal.

The summary of the invention described above is not limiting and otherand further objects, features and advantages of the invention will beapparent from the following detailed description of the presentlypreferred embodiments of the invention and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows SEAP serum concentrations at day 7 post injection of SEAPpDNA/empty DNA mixtures in the tibialis cranialis muscle of CD-1 micewith electroporation. Various SEAP pDNA amounts and empty pDNA excess(relative to the coding pDNA) were administered.

FIG. 2 shows SEAP serum concentrations at day 7 post injection of nakedSEAP pDNA or SEAP pDNA/anionic polymer mixtures in the tibialiscranialis muscle of CD-1 mice with electroporation and DNA concentrationof 2.5 micrograms in 50 microliters (half this dose per leg). Theconcentration of the anionic polymer in the injected solution varied asindicated on the graph.

FIG. 3 shows SEAP serum concentrations at day 7 post injection of nakedSEAP pDNA or SEAP pDNA/anionic polymer mixtures in the tibialiscranialis muscle of CD-1 mice with electroporation and the amount ofSEAP pDNA administered per animal was regularly (unless mentioned) 25micrograms in 50 microliters (half this dose per leg).

FIG. 4 shows SEAP serum concentrations at day 7 post injection of nakedSEAP pDNA or SEAP pDNA/anionic polymer mixtures in the gastrocnemiusmuscle of CD-1 mice and electroporation of the tissue. The concentrationof the anionic polymer in the injected solution varied as indicated onthe graph.

FIG. 5 shows SEAP serum concentrations at day 7 as a function of theamount of SEAP pDNA injected in different formulations as indicated: Ain the tibialis cranialis muscle of CD-1 mice; B in the gastrocnemiusmuscle of CD-1 mice comparing either naked SEAP pDNA or a mixture ofSEAP pDNA and a poly-L-glutamic acid at 6.0 mg/ml.

FIG. 6 shows luciferase expression after direct intramyocardialinjection of plasmid DNA formulated in saline versus poly-glutamic acid.

FIG. 7 shows hF.IX serum concentrations at day 7 post injection of nakedhF.IX pDNA or hF.IX pDNA/poly-L-glutamic acid mixtures in the tibialismuscle of C57BL/6 mice and electroporation of the tissue. Theconcentration of the anionic polymer in the injected solution varied asindicated on the graph.

FIG. 8 shows hF.IX expression in plasma of immune deficient (SCID beige)mice.

FIG. 9 depicts the immunohistology and fiber-type of hF.IX expressingmyocytes in SCID mouse muscle.

FIG. 10 A depicts plasma hF.IX levels determined by ELISA in dogsfollowing intramuscular injection of plasmid augmented byelectroporation at different numbers of sites. Values are means±SEM withn=3 for each group. FIG. 10B shows a western blot of purified hF.IXusing treated animal serum as the primary antibody. Lane A, molecularmarker; lane B, negative control serum; lane C, positive control (canineserum spiked with rabbit anti-hF.IX antibodies; lane D, serum from afemale dog from the 6 injection group (peak expression hF.IX 35.71ng/ml); lane E, serum from a male dog from the 12 injection group (peakhF.IX expression 47.9 ng/ml).

FIG. 11 depicts the duration of retention of the mouse EPO plasmid DNAfollowing delivery by electroporation using saline and poly-L-glutamicacid formulations.

FIG. 12 depicts EPO expression and hematocrit in mice following deliveryof the mouse EPO gene by electroporation using saline andpoly-L-glutamic acid formulations.

FIG. 13 depicts the results of the EPO expression in mice followingdelivery of the mouse EPO gene by electroporation using saline andpoly-L-glutamic acid formulations over a three month time frame.

FIG. 14 depicts a comparison of hINFα gene expression after delivery insaline versus polyglutamate. A depicts the results using a 50 microgramdose of plasmid DNA while B depicts the results of administration of a 5microgram dose of plasmid DNA.

FIG. 15 shows the ability of poly-L-glutamate and poloxamer formulationsto protect DNA from nuclease degradation. Panel A represents a DNA insaline formulation; Panel B represents DNA formulated in 5% PluronicF68; Panel C represents DNA formulated in 6 mg/ml poly-L-glutamate. LaneA, negative control of plasmid DNA without DNase; lane B, positivecontrol of plasmid DNA and DNase mixed 1:1; lane C, DNase diluted 1:1;lane D, DNase diluted 1:10; lane E, DNase diluted 1:100; lane F, DNasediluted 1:1,000; lane G, DNase diluted 1:10,000.

FIG. 16 depicts the results of long term biological stability of plasmidDNA encoding SEAP formulated in 6 mg/ml poly-L-glutamate under differentstorage conditions. A, lyophilization and storage at 4° C. for 105 days;B, freezing of a liquid formulation with storage at −20° C. for 105days; C, liquid storage at 4° C. for 105 days; D, liquid storage at roomtemperature for 105 days; E, liquid storage at 37° C. for 105 days; F,liquid storage at 50° C. for 8 days; G, liquid formulation subject tofreeze/thawing; H, fresh DNA formulated on poly-L-glutamate; I, freshDNA without poly-L-glutamate.

FIG. 17 depicts the plasmid map for pFN0945, an expression plasmidcarrying the gene for hF.IX. The sequence of the complete plasmid isdisclosed as SEQ. ID. NO. 3.

FIG. 18 depicts the plasmid map for pFN1645, an expression plasmidcarrying an codon optimized gene for hF.IX. The sequence of the completeplasmid is disclosed as SEQ. ID. NO. 4.

FIG. 19 depicts the plasmid map for pEP1403, an expression plasmidcarrying the mouse erythropoietin gene. The sequence of the completeplasmid is disclosed as SEQ. ID. NO. 2.

FIG. 20 depicts the plasmid map for pIF0921, an expression plasmidcarrying the human interferon alpha gene. The sequence of the completeplasmid is disclosed as SEQ. ID. NO. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The delivery and expression of sequences encoded on a vector ineukaryotic cells, particularly in vivo in a mammal, depends on a varietyof factors including transfection efficiency and lifetime of the codingsequence within the transfected cell. Thus, a number of methods arereported for accomplishing such delivery.

A non-viral gene medicine is composed of three major elements: i) anucleic acid encoding a gene product (e.g., a therapeutic protein), ii)a plasmid-based expression system, and iii) a synthetic gene deliverysystem. These products are intended to have low toxicity due to the useof synthetic components for gene delivery (minimizing for instance therisks of immunogenicity generally associated with viral vectors) andnon-integrating plasmids for gene expression. Since no integration ofplasmid sequences into host chromosomes has been reported in vivo todate, they should neither activate oncogenes nor inactivate tumorsuppressor genes. This built-in safety with non-viral systems contrastswith the risks associated with the use of most viral vectors. Asepisomal systems residing outside the chromosomes, plasmids have definedpharmacokinetics and elimination profiles, leading to a finite durationof gene expression in target tissues.

Formulating the nucleic acid with anionic polymers as disclosed below isparticularly desirable because they enhance transfection and expressionof the nucleic acid, protect the nucleic acid from degradation, and arecompletely biodegradable. In addition, because formulating the nucleicacid with anionic polymers results in more efficient transfection, loweramounts of DNA may be used. By biodegradable, it is meant that theanionic polymers can be metabolized or cleared by the organism in vivowithout any or minimal toxic effects or side effects. The term “anionicpolymers” means polymers having a repeating subunit which includes, forexample, an ionized carboxyl, phosphate or sulfate group having a netnegative charge at neutral pH. Examples of the anionic polymers includepoly-amino acids (such as poly-glutamic acid, poly-aspartic acid andcombinations thereof), poly nucleic acids, poly acrylic acid, polygalacturonic acid, and poly vinyl sulfate. In the case of polymericacids, the polymer will typically be utilized as the salt form.

Efforts have been made to enhance the delivery of plasmid DNA to cellsby physical means including electroporation, sonoporation and pressure.Injection by electroporation is a modern technique that involves theapplication of a pulsed electric field to create transient pores in thecellular membrane without causing permanent damage to the cell andthereby allows for the introduction of exogenous molecules. Thistechnique has been used widely in research laboratories to createhybridomas and is now being applied to gene transfer approaches fortherapy. By adjusting the electrical pulse generated by anelectroporetic system, nucleic acid molecules can find their way throughpassageways or pores in the cell that are created during the procedure.U.S. Pat. No. 5,704,908 describes an electroporation apparatus fordelivering molecules to cells at a selected location within a cavity inthe body of a patient. (U.S. Pat. No. 5,704,908, including any drawingscontained therein, is hereby incorporated by reference as if fully setforth herein.)

The use of electroporetic methods to deliver genes suspended in salineinto rabbit and porcine arteries as models to treat coronary andperipheral vascular disease has been discussed at the 3rd US-JapanSymposium on Drug Delivery (D. B. Dev, J. J. Giordano and D. L. Brown,Maui, Hawaii, Dec. 17-22, 1995). The ability to target and express thelacZ reporter gene suspended in saline to various depths of the dermisregion in hairless mice has been described in the article“Depth-Targeted Efficient Gene delivery and Expression in the skin byPulsed Electric Fields: An approach to Gene Therapy of Skin Aging andOther Diseases” (Zhang et al., Biochemical and Biophysical ResearchCommunications 220, 633-636 (1996)). A mammalian expression plasmid forthe lacZ gene in saline has been injected into the internal carotidartery of rats whose brain tumors had been electroporated between twoelectrodes. The gene was reported to be expressed in the tumor cellsthree days after plasmid injection and furthermore, lacZ activity wasreported to be isolated only to the tissues and cells targeted (Nishi,et al., Cancer Research 56, 1050-1055, Mar. 1, 1996).

Formulations for electroporation are described in U.S. patentapplication Ser. No. 09/322,602, which is incorporated herein byreference in its entirety, including any drawings. By adjusting theelectrical pulse generated by an electroporetic system, nucleic acidmolecules can find their way in the cell through passageways or poresthat are created during the procedure.

Previously, treatment of hemophilia B by non-viral methods was not beenpossible because only low and variable levels of gene expression wereachieved. Recently, the use of electroporation in vivo was shown toproduce consistent high levels of gene expression in muscle, liver,skin, solid tumors and testis following direct injection of plasmid intothese tissues (Titomirov, A. V., et al. (1991) Biochim Biophys Acta1088: 131-134; Muramatsu, T., et al. (1997) Biochem Biophys Res Commun233: 45-49; Suzuki, T., et al. (1998) FEBS Lett 425: 436-440; Aihara, H.and Miyazaki, J. (1998) Nat Biotechnol 16: 867-870; Mir, L. M., et al.(1998) C R Acad Sci III 321: 893-899; Rizzuto, G., et al. (1999) ProcNatl Acad Sci USA 96: 6417-6422; Goto, T., et al (2000) Proc Natl AcadSci U S A 97:354-359; Somiari, S., et al. (2000) Mol Ther 2:178-187). Inmice, electroporation of plasmid DNA in saline was used to achievecirculating levels of hF.IX that were 2% of normal and maintained for atleast 2 months (Bettan, M., et al. (2000) Mol Ther 2:204-210). Thepresent application discloses novel plasmid formulations forelectroporation that achieve four goals: (1) therapeutically significantlevels of proteins in vivo, (2) persistent expression of the transgene,(3) re-administration of formulated plasmid to obtain levels comparableto the initial levels and (4) therapeutically significant levels inlarge animals.

The delivery of a formulated DNA according to the present invention bythe use of pulse voltage delivery device represents a novel approach togene delivery. In particular, the the preferred embodiment employinganionic amino acid polymers or poly-amino acids were able tosubstantially increase the expression of introduced genes byelectroporation when compared with saline. The poly-amino acids alsohave the advantage over prior formulations by being completelybiodegradable. The preferred embodiment also provides the advantage ofallowing the uptake of formulated nucleic acid molecules (i.e., nucleicacid molecules in the compositions of the invention) by specificallytargeted cells and cell lines, as well as uptake by multiple cell linesas desired. Injecting formulated nucleic acid molecules by pulse voltagedelivery methods results in the formulated nucleic acid moleculesgaining access to the cellular interior more directly through thedestabilization of the cell wall and/or by the formation of pores as aresult of the electroporetic process. Furthermore, in certain instancesmultiple cell lines can be targeted, thus allowing contact to many morecell types than in conventional needle injection. Thus, the presentinvention provides an enhanced delivery of nucleic acid molecules andalso provides a more efficient gene delivery system which may be used togenerate an immune response, express a therapeutic gene, modulateaspects of the cell cycle or cell physiology, or provide a method toachieve other gene delivery related therapeutic methods such asanti-tumor therapy.

The term “poly-L-glutamic acid” is used interchangeably herein with“poly-L-glutamic acid, sodium salt”, “sodium poly-L-glutamate” and“poly-L-glutamate.” “Poly-L-glutamate” refers to the sodium salt ofpoly-L-glutamic acid. Although the L stereoisomer of polyglutamic acidwas found to be particularly useful, the other stereoisomer or racemicmixtures of isomers are within the scope of the invention. The presentinvention contemplates that other salts of anionic amino acid polymersmay be equally suitable.

The term “anionic amino acid polymers” means polymeric forms of a givenanionic amino acid such as, for example, poly-glutamic acid orpoly-aspartic acid. The present invention contemplates that polymersformed of a mixture of anionic amino acids, such as for example glutamicacid and aspartic acid, may be equally suitable.

By “delivery” or “delivering” is meant transportation of nucleic acidmolecules to desired cells or any cells. The nucleic acid molecules maybe delivered to multiple cell lines, including the desired target.Delivery results in the nucleic acid molecules coming in contact withthe cell surface, cell membrane, cell endosome, within the cellmembrane, nucleus or within the nucleus, or any other desired area ofthe cell from which transfection can occur within a variety of celllines which can include but are not limited to; tumor cells, epithelialcells, Langerhan cells, Langhans' cells, littoral cells, keratinocytes,dendritic cells, macrophage cells, Kupffer cells, muscle cells,lymphocytes and lymph nodes. Preferably, the composition of theinvention is delivered to the cells by electroporation and the nucleicacid molecule component is not significantly sheared upon delivery, noris cell viability directly effected by the pulse voltage deliveryprocess.

By “nucleic acid” is meant both RNA and DNA including: cDNA, genomicDNA, plasmid DNA or condensed nucleic acid, nucleic acid formulated withcationic lipids, nucleic acid formulated with peptides, cationicpolymers, RNA or mRNA. In a preferred embodiment, the nucleic acidadministered is a plasmid DNA which constitutes a “vector”. The nucleicacid can be, but is not limited to, a plasmid DNA vector with aeukaryotic promoter which expresses a protein with potential therapeuticaction, such as, for example; hGH, VEGF, EPO, IGF-I, TPO, Factor IX,IFN-α, IFN-β, IL-2, IL-12, or the like.

As used herein, the term a “plasmid” refers to a construct made up ofgenetic material (i.e., nucleic acids). It includes genetic elementsarranged such that an inserted coding sequence can be transcribed ineukaryotic cells. Also, while the plasmid may include a sequence from aviral nucleic acid, such viral sequence preferably does not cause theincorporation of the plasmid into a viral particle, and the plasmid istherefore a non-viral vector. Preferably, a plasmid is a closed circularDNA molecule. The enhancer/promoter region of an expression plasmid willdetermine the levels of expression. Most of the gene expression systemsdesigned for high levels of expression contain the intact humancytomegalovirus (CMV) immediate early enhancer/promoter sequence.However, down-regulation of the CMV promoter over time has been reportedin tissues. The hypermethylation of the CMV promoter, as observed whenincorporated into retroviral vectors, has not been observed for episomalplasmids in vivo. Nevertheless, the CMV promoter silencing could belinked to its sensitivity to reduced levels of the transcription factorNF-κB. The activity of the CMV promoter has also been shown to beattenuated by various cytokines including interferons (α and β), andtumor necrosis factor (TNF-α). In order to prolong expression in vivoand ensure specificity of expression in desired tissues, tissue-specificenhancer/promoters have been incorporated in expression plasmids. Thechicken skeletal alpha actin promoter has been shown to provide highlevels of expression (equivalent to the ones achieved with a CMV-drivenconstruct) for several weeks in non-avian striated muscles.

Additional genetic sequences in the expression plasmids can be added toinfluence the stability of the messenger RNA (mRNA) and the efficiencyof translation. The 5′ untranslated region (5′ UTR) is known to effecttranslation and it is located between the cap site and the initiationcodon. The 5′ UTR should ideally be relatively short, devoid of strongsecondary structure and upstream initiation codons, and should have aninitiation codon AUG within an optimal local context. The 5′ UTR canalso influence RNA stability, RNA processing and transcription. In orderto maximize gene expression by ensuring effective and accurate RNAsplicing, one or more introns can be included in the expression plasmidsat specific locations. The possibility of inefficient and/or inaccuratesplicing can be minimized by using synthetic introns that have idealizedsplice junction and branch point sequences that match the consensussequence. Another important sequence within a gene expression system isthe 3′ untranslated region (3′ UTR), a sequence in the mRNA that extendsfrom the stop codon to the poly(A) addition site. The 3′ UTR caninfluence mRNA stability, translation and intracellular localization.The skeletal muscle α-actin 3′ UTR has been shown to stabilize mRNA inmuscle tissues thus leading to higher levels of expression as comparedto other 3′ UTR. This 3′ UTR appears to induce a different intracellularcompartmentalization of the produced proteins, preventing the effectivetrafficking of the proteins to the secretory pathway and favoring theirperinuclear localization.

One of the attractive features of plasmid expression systems is thepossibility to express multiple genes from a single construct. Thesemultivalent systems may find applications in the expression ofheterodimeric proteins, such as antibodies, or in the in vivo productionof multiple antigens to generate a potent immune response for geneticvaccination. In cancer immunotherapy, the co-expression ofco-stimulatory molecules with a variety of cytokines may also lead toenhanced responses.

The term “vector” as used herein refers to a construction includinggenetic material designed to direct transformation of a targeted cell. Avector contains multiple genetic material, preferably contiguousfragments of DNA or RNA, positionally and sequentially oriented withother necessary elements such that the nucleic acid can be transcribedand when necessary translated in the transfected cells. The “vector”preferably is a nucleic acid molecule incorporating sequences encodingtherapeutic product(s) as well as, various regulatory elements fortranscription, translation, transcript stability, replication, and otherfunctions as are known in the art. The vector preferably allows forproduction of a product encoded for by a nucleic acid sequence containedin the vector. For example, expression of a particular growth factorprotein encoded by a particular gene. A “DNA vector” is a vector whosenative form is a DNA molecule. A “viral vector” is a vector whose nativeform is as the genomic material of a viral particle.

The term “transfection” as used herein refers to the process ofintroducing DNA (e.g., formulated DNA expression vector) into a cell,thereby, allowing cellular transformation. Following entry into thecell, the transfected DNA may: (1) recombine with that of the host; (2)replicate independently as a plasmid or temperate phage; or (3) bemaintained as an episome without replication prior to elimination.

As used herein, “transformation” relates to transient or permanentchanges in the characteristics (expressed phenotype) of a cell inducedby the uptake of a vector by that cell. Genetic material is introducedinto a cell in a form where it expresses a specific gene product oralters the expression or effect of endogenous gene products.Transformation of the cell may be associated with production of avariety of gene products including protein and RNA. These products mayfunction as intracellular or extracellular structural elements, ligands,hormones, neurotransmitters, growth regulating factors, enzymes,chemotaxins, serum proteins, receptors, carriers for small molecularweight compounds, drugs, immunomodulators, oncogenes, cytokines, tumorsuppressors, toxins, tumor antigens, antigens, antisense inhibitors,triple strand forming inhibitors, ribozymes, or as a ligand recognizingspecific structural determinants on cellular structures for the purposeof modifying their activity. This list is only an example and is notmeant to be limiting.

A “gene product” means products encoded by the vector. Examples of geneproducts include mRNA templates for translation, ribozymes, antisenseRNA, proteins, glycoproteins, lipoproteins, phosphoproteins andpolypeptides. The nucleic acid sequence encoding the gene product may beassociated with a targeting ligand to effect targeted delivery.

“Uptake” means the translocation of the vector from the extracellular tointracellular compartments. This can involve receptor-mediatedprocesses, fusion with cell membranes, endocytosis, potocytosis,pinocytosis or other translocation mechanisms. The vector may be takenup by itself or as part of a complex.

Administration as used herein refers to the route of introducing thecompositions of the invention into the body of cells or organisms.Administration includes the use of electroporetic methods as provided bya pulse voltage device to targeted areas of the mammalian body such asthe muscle cells and the lymphatic cells in regions such as the lymphnodes. Administration also includes intradermal, intra-tumoral andsubcutaneous administration.

A “therapeutically effective amount” of a composition is an amount thatis sufficient to cause at least temporary relief or improvement in asymptom or indication of a disease or condition. Thus, the amount isalso sufficient to cause a pharmacological effect. The amount of thecomposition need not cause permanent improvement or improvement of allsymptoms or indications.

The term “pulse voltage device”, or “pulse voltage injection device” asused herein relates to an apparatus that is capable of causing or causesuptake of nucleic acid molecules into the cells of an organism byemitting a localized pulse of electricity to the cells, thereby causingthe cell membrane to destabilize and result in the formation ofpassageways or pores in the cell membrane. It is understood thatconventional devices of this type are calibrated to allow one ofordinary skill in the art to select and/or adjust the desired voltageamplitude and/or the duration of pulsed voltage and therefore it isexpected that future devices that perform this function will also becalibrated in the same manner. The type of injection device is notconsidered a limiting aspect of the present invention. The primaryimportance of a pulse voltage device is, in fact, the capability of thedevice to facilitate delivery of compositions of the invention into thecells of an organism. The pulse voltage injection device can include,for example, an electroporetic apparatus as described in U.S. Pat. Nos.5,439,440, 5,704,908 or 5,702,384 or as published in PCT WO 96/12520,PCT WO 96/12006, PCT WO 95/19805, and PCT WO 97/07826, all of which areincorporated herein by reference in their entirety.

The term “apparatus” as used herein relates to the set of componentsthat upon combination allow the delivery of compositions of theinvention into the cells of an organism by pulse voltage deliverymethods. The apparatus of the invention can be a combination of asyringe or syringes, various combinations of electrodes, devices thatare useful for target selection by means such as optical fibers andvideo monitoring, and a generator for producing voltage pulses which canbe calibrated for various voltage amplitudes, durations and cycles. Thesyringe can be of a variety of sizes and can be selected to injectcompositions of the invention at different delivery depths such as tothe skin of an organism such as a mammal, or through the skin.

The term “organism” as used herein refers to common usage by one ofordinary skill in the art. The organism can include microorganisms, suchas yeast or bacteria, plants, birds, reptiles, fish or mammals. Theorganism can be a companion animal or a domestic animal. Preferably theorganism is a mammal and is therefore any warmblooded organism. Morepreferably the mammal is a human.

The term “companion animal” as used herein refers to those animalstraditionally treated as “pets” such as for example, dogs, cats, horses,birds, reptiles, mice, rabbits, hamsters, and the like. The term“domestic animal” as used herein refers to those animals traditionallyconsidered domesticated, where animals such as those considered“companion animals” are included along with animals such as, pigs,chickens, ducks, cows, goats, lambs, and the like.

By “prolong the localized bioavailability of a nucleic acid” is meantthat a nucleic acid when administered to an organism in a compositioncomprising such a compound will be available for uptake by cells for alonger period of time than if administered in a composition without sucha compound, for example when administered in a formulation such as asaline solution. This increased availability of nucleic acid to cellscould occur, for example, due to increased duration of contact betweenthe composition containing the nucleic acid and a cell or due toprotection of the nucleic acid from attack by nucleases. The compoundsthat prolong the localized bioavailability of a nucleic acid aresuitable for internal administration.

By “suitable for internal administration” is meant that the compoundsare suitable to be administered within the tissue of an organism, forexample within a muscle or within a joint space, intradermally orsubcutaneously. Other forms of administration which may be utilized aretopical, oral, pulmonary, nasal and mucosal; for example, buccal,vaginal or rectal. Properties making a compound suitable for internaladministration can include, for example, the absence of a high level oftoxicity to the organism as a whole.

By “solutions” is meant water soluble polymers and/or surfactants insolution with nucleic acids.

Polymeric Formulations for Plasmid Delivery to Muscle

The present invention provides polymeric formulations that addressproblems associated with injection of nucleic acids suspended in saline.Unformulated (naked nucleic acid molecules) plasmids suspended in salinehave poor bioavailability in muscle due to rapid degradation of plasmidby extracellular nucleases. One possible approach to overcome the poorbioavailability is to protect plasmid from rapid nuclease degradationby, for example, condensing the plasmid with commonly used cationiccomplexing agents. However, due to the physiology of the muscle, the useof rigid condensed particles containing plasmid for efficienttransfection of a larger number of muscle cells has not been successfulto date. Cationic lipid and polylysine plasmid complexes do not crossthe external lamina to gain access to the caveolae and T tubules (Wolff,J. A., et al., 1992, J. Cell. Sci. 103:1249-1259).

Thus, the invention increases the bioavailability of plasmid in muscleby: protecting plasmid from rapid extracellular nuclease degradation;dispersing and retaining intact plasmid in the muscle and/or tumor; andfacilitating the uptake of plasmid by muscle and/or tumor cells. Aspecific method of accomplishing this, which preferably is used inconjunction with pulse voltage delivery, is the use of anionic polymers.

Administration

Administration as used herein refers to the route of introduction of aplasmid or carrier of DNA into the body. Administration can be directlyto a target tissue or by targeted delivery to the target tissue aftersystemic administration. In particular, the present invention can beused for treating conditions by administration of the formulation to thebody in order to establish controlled expression of any specific nucleicacid sequence within tissues at certain levels that are useful for genetherapy.

The preferred means for administration of vector (plasmid) and use offormulations for delivery are described above. The preferred embodimentsare by pulse voltage delivery to cells in combination with needle orneedle free injection, or by direct applied pulse voltage wherein theelectroporation device's electrodes are pressed directly against thetargeted tissue or cells, such as for example epidermal cells, and thevector is applied topically before or after pulse application anddelivered through and or to the cells.

The route of administration of any selected vector construct will dependon the particular use for the expression vectors. In general, a specificformulation for each vector construct used will focus on vector deliverywith regard to the particular targeted tissue, the pulse voltagedelivery parameters, followed by demonstration of efficacy. Deliverystudies will include uptake assays to evaluate cellular uptake of thevectors and expression of the DNA of choice. Such assays will alsodetermine the localization of the target DNA after uptake, andestablishing the requirements for maintenance of steady-stateconcentrations of expressed protein. Efficacy and cytotoxicity can thenbe tested. Toxicity will not only include cell viability but also cellfunction.

Muscle cells have the unique ability to take up DNA from theextracellular space after simple injection of DNA particles as asolution, suspension, or colloid into the muscle. Expression of DNA bythis method can be sustained for several months.

The chosen method of delivery should result in expression of the geneproduct encoded within the nucleic acid cassette at levels that exert anappropriate biological effect. The rate of expression will depend uponthe disease, the pharmacokinetics of the vector and gene product, andthe route of administration, but should be in the range 0.001-100 mg/kgof body weight/day, and preferably 0.01-10 mg/kg of body weight/day.This level is readily determinable by standard methods. It could be moreor less depending on the optimal dosing. The duration of treatment willextend through the course of the disease symptoms, possiblycontinuously. The number of doses will depend upon the disease, deliveryvehicle, and efficacy data from clinical trials.

DNA Injection Variables

The level of gene delivery and expression or the intensity of an immuneresponse achieved with the present invention can be optimized byaltering the following variables. The variables are: the formulation(composition, plasmid topology), the technique and protocol forinjection (area of injection, duration and amplitude of voltage,electrode gap, number of pulses emitted, type of needle arrangement,pre-injection-pulsed or post-injection-pulsed cells, state of muscle,state of the tumor), and, the pretreatment of the muscle with myotoxicagents. An immune response can be measured by, but is not limited to,the amount of antibodies produced for a protein encoded and expressed bythe injected nucleic acid molecule.

Other injection variables that can be used to significantly affect thelevels of proteins, antibodies and/or cytotoxic T-lymphocytes producedin response to the protein encoded by the formulated nucleic acidmolecule provided by the pulse voltage injection method of the presentinvention are the state of the muscle being injected and injectiontechnique. Examples of the variables include muscle stimulation, musclecontraction, muscle massage, delivery angle, and apparatus manipulation.Massaging the muscle may force plasmid out of the muscle either directlyor via lymphatic drainage. By altering the depth of penetration and/orthe angle at which the pulse voltage device is placed in relation tomuscle fibers the present invention improves the plasmid distributionthroughout the injection area that subsequently increases the antibodyresponse to the protein which is encoded and expressed by the plasmid.

Nucleic Acid Based Therapy

The present invention can be used to deliver nucleic acid vaccines in amore efficient manner than is conventionally done at the present time.Nucleic acid vaccines, or the use of plasmid encoding antigens ortherapeutic molecules such as Human Growth Hormone, has become an areaof intensive research and development in the last half decade.Comprehensive reviews on nucleic acid based vaccines have been published(M. A. Liu, et al. (Eds.), 1995, DNA Vaccines: A new era in vaccinology,Vol. 772, Ann. NY. Acad. Sci., New York; Kumar, V., and Sercarz, E.,1996, Nat. Med. 2:857-859; Ulmer, J. B., et al., (Eds.) Current Opinionin Immunology; 8:531-536. Vol. 772, Ann. NY. Acad. Sci., New York).Protective immunity in an animal model using plasmid encoding a viralprotein was first observed in 1993 by Ulmer et al. (Ulmer, J. B., etal., 1993, Science 259:1745-1749). Since then, several studies havedemonstrated protective immunity for several disease targets and humanclinical trials have been started.

Many disease targets have been investigated. Examples include antigensof Borrelia burgdorferi, the tick-borne infectious agent for Lymedisease (Luke et al., J. Infect. Dis. 175:91-97, 1997), humanimmunodeficiency virus-1, (Letvin et al., Proc. Nat. Acad. Sci. USA94:9378-9383, 1997), B cell lymphoma (Syrengelas et al., NatureMedicine. 2:1038-41, 1996), Herpes simplex virus (Bourne et al., J.Infectious dis. 173:800-807, 1996), hepatitis C virus (Tedeschi et al.,Hepatology 25:459-462, 1997), rabies virus (Xiang et al., Virology,209:569-579, 1995), Mycobacterium tuberculosis (Lowrie in GeneticVaccines and Immunotherapeutic Strategies CAThibeault, ed. Intl BusComm, Inc., Southborough, Mass. 01772 pp. 87-122, 1996), and Plasmodiumfalciparum (Hoffman et al., Vaccine 15:842-845, 1997). Additionally,nucleic acid based treatment for reducing tumor-cell immunogenicity,growth, and proliferation is indicative of gene therapy for diseasessuch as tumorigenic brain cancer (Fakhrai et al., Proc. Natl. Acad.Sci., 93:2909-2914, 1996).

An important goal of gene therapy is to affect the uptake of nucleicacid by cells, thereby causing an immune response to the protein encodedby the injected nucleic acid. Nucleic acid based vaccines are anattractive alternative vaccination strategy to subunit vaccines,purified viral protein vaccines, or viral vector vaccines. Each of thetraditional approaches has limitations that are overcome if theantigen(s) is expressed directly in cells of the body. Furthermore,these traditional vaccines are only protective in a strain-specificfashion. Thus, it is very difficult, and even impossible usingtraditional vaccine approaches to obtain long lasting immunity toviruses that have several sera types or viruses that are prone tomutation.

Nucleic acid based vaccines offer the potential to produce long lastingimmunity against viral epitopes that are highly conserved, such as withthe nucleoprotein of viruses. Injecting plasmids encoding specificproteins by the present invention results in increased immune responses,as measured by antibody production. Thus, the present invention includesnew methods of providing nucleic acid vaccines by delivering aformulated nucleic acid molecule with a pulse voltage device asdescribed herein.

The efficacy of nucleic acid vaccines is enhanced by one of at leastthree methods: (1) the use of delivery systems to increase the stabilityand distribution of plasmid within the muscle, (2) by the expression (ordelivery) of molecules to stimulate antigen presentation/transfer, or(3) by the use of adjuvants that may modulate the immune response.

Diseases and Conditions for Intramuscular Plasmid Delivery

The present invention described herein can be utilized for the deliveryand expression of many different coding sequences. The coding sequencesmay be used to ameliorate the effects of inborn errors of metabolism,genetic deficiencies of certain necessary proteins, acquired metabolicand regulatory imbalances and disordered cellular regulation such aswith cancer. The coding sequence containing composition preferably isadministered by pulsed voltage delivery and may require, as needed,exposure of the tissue to be treated by surgical means as determined bya certified professional.

EXAMPLES

The following examples are offered by way of illustration and are notintended to limit the scope of the invention in any manner. One ofordinary skill in the art would recognize that the various moleculesand/or amounts disclosed in the examples could be adjusted orsubstituted. It would also be recognized that the delivery targetsand/or amounts delivered in the examples could be adjusted orsubstituted by selecting different muscles for injection, injection intotumors or nodes, or increasing or decreasing the duration of pulse timeor alternating the pulse application from pre-injection topost-injection.

Preparation of Formulations

Formulations were made by aliquoting appropriate volumes of sterilestock solutions of water, plasmid, polymer, buffer and/or 5M NaCl toobtain a final plasmid in an isotonic solution. The total plasmidconcentration of all formulations was measured by UV absorption at 260nm. The osmotic pressure of selected formulations was measured using aFiske One-Ten Micro-Sample Osmometer (Fiske Associates; Norwood, Mass.).The percentage of supercoiled plasmid was measured using 1% agarose gelelectrophoresis followed by fluorimaging.

Plasmids were formulated in 5-10 mM Tris, pH 7.5 or saline (150 mM NaCl)or mixed with a polymer in isotonic saline. Plasmid used for injectionwas formulated with various polymers in an isotonic saline solution.Typically, the concentration of plasmid was 1-2 mg/ml in saline, orformulated with polyvinylpyrrolidone (PVP, 5%) or 6 mg/mlpoly-L-glutamate (Sigma, St Louis, Mo.) in saline.

Anionic polymers included poly-L-glutamic acid (p-L-Glu), sodium salt,of various molecular weights (degree of polymerization (DP) of 9 (SigmaP1943), degree of polymerization of 10 (Sigma P1818),2-15 kDa (SigmaP4636),15-50 kDa (Sigma P4761) and 50-100 kDa (Sigma P4886)),poly-D-glutamic acids (p-D-Glu) of 15-50 (Sigma P4033) and 50-100 kDa(Sigma 4637), poly-L-aspartic acid (p-L-Asp), sodium salt, of 2-15(Sigma P5387) and 15-50 kDa (Sigma P6762) and poly-acrylic acid (pAA),sodium salt, of 5 and 60 kDa. The polyamino acids were purchased fromSigma (St. Louis, Mo.), while the poly(acrylic acid) was acquired fromFluka (Switzerland).

The DNA/anionic polymer formulations were preferably prepared byaliquoting appropriate volumes of sterile stock solutions of plasmid,anionic polymer and 5M NaCl to obtain selected final plasmid and anionicpolymer concentrations. The anionic polymer was added to the DNAsolution prior to adding salt for tonicity adjustment. Thus,poly-L-glutamate formulations are preferably prepared by combining anaqueous stock solution of sodium poly-L-glutamate (sodium salt ofpoly-L-glutamic acid) with a stock solution of purified plasmid DNA insaline or up to 10 mM Tris, pH 7.5. After the poly-L-glutamic acid andDNA are combined, the solution is adjusted to a final concentration of150 mM NaCl by addition of a stock solution of 5M NaCl.

The osmolality of each formulation was measured using a Fiske One-TenMicro-Sample Osmometer (Fiske Associate, Norwood Mass.). Formulationswere also characterized by measuring the optimal density at 260 and 280nm, and by determining plasmid conformation on a 1% agarose gel.

Stability Test for Plasmid in the Formulation

For the analysis of pDNA stability in the formulation, 50 ng offormulated pDNA with 5 microliters of tracking dye was loaded into 1%agarose gel in 1% tris-acetate-EDTA (TAE) buffer and run the gel at 100volts for 1-2 hours. The gel was then stained with SYBR Green II(Molecular Probes, Inc.) for 20 minutes. The stained gel was washed withwater and % of supercoiled and open circled DNA was determined using aFluorinate (Molecular Dynamics Co., Sunnyvale, Calif.).

Elisa Protocol

High affinity assay plates were coated with antigen diluted in PBS (50microliters/well) and placed at 4° C. overnight. After allowing plate(s)to come to room temperature, all wells were blocked with 200microliters/well of 4% BSA/4% NGS solution made in 1× PBS/Tween20 for 1hr at 37° C. Add serum samples (50 microliters/well at a startingdilution of 1:100 in 4% BSA/4% NGS/PBS/Tween20, in duplicate) andincubate for 1-2 hours at 37° C. Wash plate(s) with PBS/Tween 20 and add50 microliters/well of HRP-conjugated secondary, diluted in 1% BSA, andincubate at 37° C. for 1 hour. Wash plate(s) with PBS/Tween 20 and add100 microliters/well of TMB soluble reagent. Incubate at roomtemperature for 10 minutes and stop the reaction by adding 50microliters/well of 0.2M H₂SO₄. Read plate(s) at 450 nm.

Plasmids

Plasmids pAP1166 and pFN0945 (SEQ. ID. NO. 3) containing a CMVenhancer-promoter and either a human placental secreted alkalinephosphatase reporter gene (SEAP) (pAP1166) or the coding region of hF.IX(pFN0945 SEQ. ID. NO. 3) were manufactured and purified at Valentis,Inc. The plasmid map of pFN0945 is shown in FIG. 17. Human factor IX(hF.IX) plasmid was prepared by inserting a synthetic coding sequence inwhich rare codons were converted to prevalent ones and potential crypticsplice sites were abrogated (Oberon Technologies Inc., Alameda, Calif.).The hF.IX coding sequence was inserted into the Valentis plasmidbackbone containing a 107 bp 5′TR, a 117 bp synthetic intron, the humangrowth hormone polyadenylation signal, a PUC12 origin of replication anda kanamycin resistance gene. The hF.IX gene was driven by the CMVenhancer/promoter. Plasmids were grown in Escherichia coli DH5α and werepurified using a proprietary method involving alkaline lysis andchromographic methods (Abruzzese, R. V., et al. (1999) Hum Gene Ther10:1499-1507, incorporated herein by reference). The human secretedalkaline phosphatase (SEAP) and human erythropoietin plasmids wereidentical to the hF.IX plasmid except for the coding region.

Experimental Animals

Male C57BL/6 mice (19-21 g), male CD-1 mice (29-31 g), maleC.B-17/lcrCrl-scid-bgBR (SCID BEIGE) mice (7 weeks of age) and femaleC57BL/6 mice (7-8 weeks) were obtained from Charles River Laboratoriesand were acclimatized for a 3-7 day period in a 12 hour light-dark cycleat 23° C./40% RH in accordance with state and federal guidelines. Food(Purina rodent chow) and water were provided ad libitum. The animalswere housed in hepa-filtered caging units (4 mice per isolator) withsterilized bedding food and water. Cage exchange and all manipulationswith the SCID mice were performed in a laminar flow hood. Animals wereanesthetized via intraperitioneal (IP) injection with a combinationanesthesia (Ketamine, Xylazine and Acepromazine) at a dose of 1.8-2.0mL/kg (mice). Beagle dogs (Harlan, Indianapolis, Ind.) were maintainedat Stillmeadow, Inc. (Sugarland, Tex.) in accordance with the guidelinesof the Institutional Animal Care and Use Committee.

Animal Injections

After anesthesia, hind limbs were shaved and scrubbed with betadinefollowed by 70% ethanol. 10 microliters of the formulation was injectedwith 10 micrograms of formulated plasmid using a 0.3-ml insulin syringewith a 28-gauge, 0.5 needle (Becton Dickinson, Granklin Lake, N.J.). Theinjected volumes in mice were 25 microliters and 50 microliters in thecranial tibialis and gastrocnemius, respectively. Where indicated, sevendays after formulation injection, the animals were sacrificed by CO₂asphyxiation and the tibialis anterior muscles was harvested, quicklyimmersed in liquid nitrogen, and lyophilized overnight. The driedmuscles were used or stored at −80° C. for further determination ofreporter gene activity.

Device and Dosing Regimens

Plasmid formulated at the required dose was administered in rodents bylongitudinal injection in both tibialis cranialis or in bothgastrocnemius muscles (bilateral administration). By holding the entirelower leg between the caliper electrodes good “electrotransfection”could be obtained. Approximately, two minutes after injection, anelectric field was applied in the form of 2 square wave pulses (one persecond) of 25 millisecond (“ms”) each and 375 V/cm delivered by anElectro Square Porator (T820, BTX, San Diego, Calif.). The clampelectrodes consist of 2 stainless steel parallel plate calipers (1.5 cm²) that are placed in contact with the skin so that the leg is held in asemi-extended position throughout pulse administration. The separationdistance of the electrodes is described. Typically the leg of the mousewas positioned between the two plates, which were compressed togetheruntil snug with a 3-4 mm separation distance between the plates. Two 25ms pulses at a voltage of 375 V/cm were then generated with a T-820Electro Square Porator (Genetronics, San Diego, Calif.). The pulses wereadministered at a rate of ˜1/second.

Dogs were anesthetized with isofluorane for the injection andelectroporation procedures. A 6-needle array electrode was used(Genetronics, San Diego, Calif.) (Jaroszeski, M. J., et al. (1997)Biochim Biophys Acta 1334:15-18). The electroporation regimen was 6pulses of 60 ms duration at a voltage of 200 V/cm. The polarity of thepulse was reversed following each pulse under the control of an AutoSwitcher (Genetronics, San Diego, Calif.). Following the electroporationprocedure the skin above injected muscle was tattooed to identify theinjection site for later analysis. Carbon particles were also injectedin some of the muscles following electroporation as a marker of theinjection site for histological analyses.

In one embodiment, the gene delivery approach uses a low voltage (375V/cm), long pulse (25 ms) electroporation regimen in mice, in contrastto other protocols that use high voltage (1,800 V/cm) and short pulse(100 μs) parameters (Vicat, J. M., et al (2000) Hum Gene Ther11:909-916).

Serum Assays

Blood samples were collected at the appropriate time points followingplasmid administration. Mice were anesthetized IP with Ketamine (60mg/kg) (Phoenix Scientifics, Inc., St Louis, Mo.). A proparacainehydrochloride opthalmic solution (Solvay Animal Health Inc., MendotaHeights, Minn.) was applied to the eye. The blood was collected inMicrotainer® serum separator tubes (Becton Dickinson, Franklin Lakes,N.J.) and allowed to clot for 15-30 minutes before centrifuging at 7,000rpm for 5 minutes. Serum levels of SEAP were determined using achemiluminescence assay (Tropix, Bedford, Mass.) following themanufacturers instructions.

For F.IX assays, blood samples were obtained from the retro-orbitalplexus of mice. Approximately 250 microliters of blood were collected inEDTA microtainer tubes (Becton Dickinson, Franklin Lakes, N.J.). Theblood was centrifuged at ˜5,000 g for 5 minutes. Plasma samples werefrozen at −80° C. and stored until used for analysis. Plasma hF.IXlevels were determined using the Asserachrom IX:Ag human F.IX ELISA kit(Diagnostica Stago, France). Purified human F.IX (Sigma, St. Louis, Mo.)was used to generate a standard curve. For dogs, blood was collectedfrom the jugular vein of conscious animals into EDTA plasma tubes.Reference plasma for the ELISAs was obtained from each animal prior totreatment. Serum levels of erythropoietin were determined using acommercially available ELISA kit from R&D Systems (Minneapolis, Minn.).

Western Blot Analysis

Purified hF.IX (Sigma, St. Louis, Mo.) in sample buffer (0.5 M Tris,1.5% SDS, 4% β-mercaptoethanol, 10% glycerol, 0.03% bromphenol blue) wasloaded on a 10% glycine Tris polyacrylamide gel (Novex, San Diego,Calif.). Following electrophoresis, protein was transferred to anitrocellulose membrane (Novex, San Diego, Calif.). The membranes werethen incubated first in canine plasma (1:50) from either treated animalsor normal dogs (negative control). For the positive control the membranewas incubated in normal canine plasma spiked with rabbit anti-hF.IXantibody (1:1,000 final). The second antibody was either horseradishperoxidase (HRP)-conjugated rabbit anti-canine antibody (Sigma, St.Louis, Mo.) or HRP conjugated sheep anti-rabbit antibody (Sigma, St.Louis, Mo.). Bands on the blots were visualized using a peroxidasesubstrate kit (Vector Laboratories Inc., Burlingame, Calif.).

Creatine Kinase (CK)

Serum collected from the dogs was frozen and shipped on dry ice byovernight courier to IDEXX Veterinary Services (West Sacramento, Calif.)for analysis of CK levels by standard methodology.

Histological Analysis and Fiber-Typing

For hF.IX immunohistochemistry in mouse tissue a method modified fromHerzog et al. (1997) Proc. Natl. Acad. Sci. USA 94(11), 5804-5809, wasused. Briefly, 10 micrometer cryosections of tissue were fixed in 3%paraformaldehyde for 15 minutes, rinsed in PBS, treated with methanolfor 10 minutes, washed three times in PBS and then blocked in 20% normalgoat serum. Sections were subsequently incubated for 1 hour with anaffinity-purified rabbit anti-hF.IX (Dako Corp., Carpinteria, Calif.)that was diluted 1:6,000 in PBS/1% BSA. The sections were rinsed PBS andincubated with biotinylated goat anti-rabbit IgG (Vector Laboratories,Burlingame, Calif.) diluted 1:400 in PBS for 30 minutes. The sectionswere rinsed and hF.IX staining was visualized using the Elite ABCreagent (Vector Laboratories, Burlingame, Calif.) at a dilution of 1:80for 30 minutes followed by a 5 minute incubation in a DAB solution(Vector Laboratories, Burlingame, Calif.). The sections werecounterstained with Mayer's hematoxylin (VWR, Houston, Tex.). Allincubation steps were at room temperature.

For ATPase fiber subtyping, 10 micrometers of muscle tissue cryosections(serial sections of those used for the hF.IX staining) were incubatedfor 5 minutes in barbital acetate buffer, pH 4.6, transferred to ATPasesolution, pH 9.4, for 20 minutes, washed three times in 1% calciumchloride, washed for 5 minutes in 2% cobalt chloride, washed ten timesin 0.01 M sodium barbital wash solution, and rinsed in distilled waterfor 5 minutes. To visualize the ATPase activity, sections were dippedinto 1.5% ammonium sulfide for 20 seconds, rinsed in distilled water,dehydrated in ethanol, and coverslipped. At pH 4.6, type I fibers staindark brown, type IIA fibers stain very light brown and type IIB fibersare intermediate.

For dogs, muscle samples were harvested and immediately placed in 10%neutral buffered formalin overnight at room temperature. The tissue wasdehydrated using alcohol and then embedded in paraffin. Sections werecut and stained with Mayer's hematoxylin and eosin (Sigma, St. Louis,Mo.).

All microscopy was performed with an Olympus BX-40 (Olympus America,Melville, N.Y.) microscope equipped with a DXC-960MD color video camera(Sony Corp., Japan).

Example I Determination of Formulation and Delivery Parameters UsingReporter Genes

Formulating DNA with anionic polymers increases electroporation-mediatedgene expression after an intra-muscular injection. An example of ananionic polymer is an excess of non-coding DNA, which can increasetransgene expression. The protocol that was regularly used to transfectthe myofibers of CD-1 or C57BL/6 mice consisted of an injection of a DNAsolution followed, two minutes later, by the electroporation of theinjected muscle with a clamp electrode. A constant mass (0.75micrograms, 2.5 micrograms or 15 micrograms) of a plasmid DNA coding forthe SEAP (human placental secreted alkaline phosphatase) gene withvarious amounts of an empty plasmid was co-injected in the tibialiscranialis muscle of CD-1 mice. Empty plasmid means that the plasmid doesnot carry the coding sequences for SEAP or, preferably, any other gene.

FIG. 1 shows SEAP serum concentrations at day 7 post injection of SEAPpDNA/empty DNA mixtures in the tibialis cranialis muscle of CD-1 miceand electroporation of the tissue. Various SEAP pDNA amounts (0.15micrograms, 0.75 micrograms, 2.5 micrograms, 6.25 micrograms and 15micrograms) and empty pDNA excess (relative to the coding pDNA) wereadministered in 50 microliters per animal (half this dose per leg). Foreach dose of SEAP pDNA tested, SEAP concentration in the serum at thepeak of expression (day 7 post injection/electroporation) increasedsubstantially when a 2-fold excess of empty pDNA was co-administeredwith the coding pDNA. For instance, SEAP expression in these conditionswith 2.5 micrograms SEAP pDNA was similar to that obtained with 6.25micrograms SEAP pDNA without an empty plasmid. When the amount of SEAPpDNA administered was 2.5 or 15 micrograms, increasing further theexcess of empty vector (6, 30 and 120-fold) resulted in a continuousdecrease of SEAP expression. Conversely, for the lowest amount of codingpDNA (0.75 micrograms), SEAP expression was maintained when a 6-foldexcess of empty DNA was co-injected.

This non-monotonous evolution of SEAP expression as the amount of emptyDNA pre-mixed with the SEAP pDNA is increased reflects the interplay oftwo phenomena. First, the addition of the empty pDNA enhances geneexpression due to the saturation of a DNA degradation mechanism or thesaturation of a process that deactivates the DNA (e.g., binding tocationic entities such as divalent cations or histones, in theinterstitial fluid and in the myocytes nuclei, respectively). Thiseffect can result either in an increased intracellular (or intranuclear)uptake or in a more efficient processing of the SEAP pDNA in thenucleus. Second, the empty vector competes with the SEAP-coding DNA insome of the steps that leads to transcription of the transgene, whichresults in a decrease of SEAP expression. These steps include thedistribution of the DNA in the interstitial fluid prior toelectroporation, the intracellular entry through the electropores, thetrafficking to the nuclei, the entry in the nuclei and the binding totranscription factors.

Thus, polynucleotides having non-coding sequences or preferably randomsequences may function to protect against degradation in vivo of plasmidcarrying a gene intended to be expressed in an animal.

In addition to using polynucleotides or empty plasmid to enhancetransgene expression and protect against degradation, other anionicpolymers may also be used. These anionic polymers may include poly-aminoacids (such as poly-L-glutamic acid, poly-D-glutamic acids,poly-L-aspartic acid, poly-D-aspartic, and combination thereof) orpoly-organic acids (such as poly-acrylic acid) which exhibit beneficiaryeffects similar to the empty plasmid, but which do not compete with theSEAP pDNA in the processes described above.

Some anionic polymers were found to be considerably more potent thannon-coding DNA to increase transgene expression. Anionic polymers withvarious origins, molecular weights, conformations and charge densitieswere mixed at various concentrations with the SEAP pDNA (0.05 mg/ml)prior to injection in the tibialis cranialis muscle of CD-1 mice. Sevendays after the injection/electroporation procedure (at the peak ofexpression), SEAP serum concentrations were determined (FIG. 2). At thelow DNA dose tested (1.25 micrograms per tibialis), some of the anionicpolymers selected considerably increased SEAP expression. The highestSEAP levels were obtained with the 60 kDa poly-acrylic acid (pAA) at 3.0mg/ml and the 2-15 kDa poly-L-glutamic acid at 6.0 mg/ml.Co-administration of these anionic polymers with the SEAP pDNA enhancedexpression by 10 and 8-fold, respectively (FIG. 2).

In order to characterize further the beneficiary effect provided by theanionic polymers, the same type of experiment as that mentioned abovewas carried out, but at a 10-fold higher DNA concentration of 0.5 mg/ml.FIG. 2 shows SEAP serum concentrations at day 7 post injection of nakedSEAP pDNA or SEAP pDNA/anionic polymer mixtures in the tibialiscranialis muscle of CD-1 mice and electroporation of the tissue. Theamount of SEAP pDNA administered per animal was 2.5 micrograms in 50microliters (half this dose per leg). The concentration of the anionicpolymer in the injected solution varied as indicated on the graph. FIG.3 shows the same thing as FIG. 2, except that the amount of SEAP pDNAadministered per animal was regularly (unless mentioned) 25 microgramsin 50 microliters (half this dose per leg). The concentration of theanionic polymer (or anionic monomer when applicable) in the injectedsolution varied as indicated on the graph.

At this high DNA concentration, the range of enhancements in SEAPexpression resulting from the addition of an anionic polymer was lowerthan that observed previously (FIG. 2, 3). In particular, thepoly-acrylic acids, highly efficient at a low DNA dose, were almostinactive. However, the polypeptides still increased SEAP expressionsubstantially (up to 2-fold with the 2-15 kDa poly-L-glutamic acid at6.0 mg/ml). This result was particularly remarkable given that SEAPexpression was reaching a plateau at this concentration of DNA. Indeed,when the DNA was administered “naked”, SEAP expression was enhanced byonly 50% and 15% following an increase in DNA concentration by 3-fold(from 0.5 mg/ml to 1.5 mg/ml) and 10-fold (to 5.0 mg/ml), respectively(FIG. 3).

The fact that the L-glutamic acid monomer was unable to increaseexpression, in contrast to the 2-15 kDa polymer (FIG. 3), demonstratedthat a macromolecule is necessary to provide the effect that leads tohigher expressions. When the results from the two separate experimentspartially displayed in FIG. 2 and FIG. 3 are gathered in the compositegraph (FIG. 5A), the evolution of SEAP expression as a function of DNAconcentration can be compared for the naked DNA injection and two of theDNA/anionic polymers treatments (namely DNA/2-15 kDa poly-L-glutamicacid at 6.0 mg/ml and DNA/60 kDa poly-acrylic acid (pAA) at 3.0 mg/ml).Two different trends appear clearly after adding an anionic polymer tothe DNA solution. In the case of the 60 kDa poly-acrylic acid, theincrease in SEAP expression (compared to naked DNA) is high butrestricted to low and intermediate DNA concentrations. In the case ofthe 2-15 kDa poly-L-glutamic acid, the levels of expression are slightlylower in this range of DNA concentrations, but the beneficiary effect isstill substantial at high DNA concentrations.

The injection/electroporation procedure was conducted in thegastrocnemius muscle of CD-1 mice, instead of the tibialis cranialis, todetermine if the increase in expression provided by some anionicpolymers is specific to the muscle used for expression. The anionicpolymers selected were those that yielded the highest levels ofexpression in the studies described above, i.e., the 2-15 kDa and 50-100kDa poly-L-glutamic acids as well as the 60 kDa poly-acrylic acid. TwoDNA concentrations were tested in this study, i.e., 0.3 mg/ml (15micrograms injected per gastrocnemius) and 1 mg/ml. FIG. 4 shows SEAPserum concentrations at day 7 post injection of naked SEAP pDNA or SEAPpDNA/anionic polymer mixtures in the gastrocnemius muscle of CD-1 miceand electroporation of the tissue. The amount of SEAP pDNA administeredper animal was either 30 micrograms, 100 micrograms or 300 micrograms in100 microliters (half this dose per leg). The concentration of theanionic polymer in the injected solution varied as indicated on thegraph.

The three polymers yielded a substantial increase in expression at thelow DNA dose (FIG. 4). Conversely to what was observed when theinjections were performed in the tibialis cranialis muscle, the 60 kDapoly-acrylic acid was most efficient at its lowest concentration of 0.6mg/ml and was less potent than the poly-L-glutamic acids used at 6.0 or12.0 mg/ml. In the best conditions tested (50-100 kDa poly-L-glutamicacid at 6.0 mg/ml), SEAP expression was increased by 8-fold over thatobtained with naked DNA. At the higher DNA concentration, the trendsdescribed above were accentuated. The 60 kDa poly-acrylic acid waseither inactive or inhibitory at high concentrations, whereas thepoly-L-glutamic acids were still yielding a 2 to 3-fold increase inexpression. Again, this result was particularly remarkable, given thatthe expression levels achieved with the naked DNA treatment were onlyincreased by 10% when the DNA concentration was elevated to 3.0 mg/mlinstead of 1.0 mg/ml.

FIG. 5A shows SEAP serum concentrations at day 7 as a function of theamount of SEAP pDNA injected in the tibialis cranialis muscle of CD-1mice. Solutions administered two minutes before electroporationconsisted of either naked SEAP pDNA or a mixture of SEAP pDNA and a 60kDa poly-acrylic acid at 3.0 mg/ml or a mixture of SEAP pDNA and a 2-15kDa poly-L-glutamic acid at 6.0 mg/ml. FIG. 5B shows SEAP serumconcentrations at day 7 as a function of the amount of SEAP pDNAinjected in the gastrocnemius muscle of CD-1 mice. Solutionsadministered two minutes before electroporation consisted of eithernaked SEAP pDNA or a mixture of SEAP pDNA and a poly-L-glutamic acid at6.0 mg/ml. When the SEAP serum concentration at day 7 post-injection isplotted as a function of the amount of DNA injected per animal as inFIG. 5B, the beneficiary effect of the poly-L-glutamic acids (at 6.0mg/ml) on expression appears clearly.

Example II Determination of Reporter Gene Expression Using Poly-GlutamicAcid without Electroporation

In order to determine the ability of sodium poly-glutamate to increasethe expression of genes encoded on plasmid DNA without electroporation,plasmid DNA formulated in saline was compared with a formulation insodium poly-glutamate for expression after direct intramyocardialinjection in mice.

Plasmid DNA encoding luciferase (pLC0888) was formulated in saline or 6%sodium poly-L-glutamate ((Sigma P4636) at plasmid concentrations of 1and 3 mg/mL. A total of twenty CD-1 male mice (29-31g) were used. Themyocardium was injected directly after surgical exposure. Ten (10)microliters of formulation (using a 3/10 cc insulin syringe) wereinjected into the apex of the heart (i.e., left ventricle). The heartwas repositioned and the thorax sutured. Seven days after injection, thehearts were removed and snap frozen in liquid nitrogen, and stored at−80° C. until needed for analysis. For analysis, heart muscle wasbead-beat for 2 minutes prior to addition of 1 milliliter of 0.5× Lysisbuffer. The tissue was bead-beat for 5 minutes and centrifuged for 10mins at 13,000 rpm. The supernatants were assayed for luciferaseactivity. The results of luciferase expression at 7 days after injectionare shown in FIG. 6. Each bar represents n=5. As shown in FIG. 6,plasmid DNA formulated with poly-L-glutamate increased gene expressionseveral fold over saline.

Example III Expression of Therapeutic Genes Factor IX Expression UsingPolymer Formulations

In addition to reporter genes, experiments were also performed usingpoly-L-glutamic acids to increase the expression of a therapeutic gene,namely that coding for the coagulation factor IX. The potency of theseanionic polymers was tested with pFN0945 (SEQ. ID. NO. 3 and FIG. 17) atDNA concentrations (0.5 mg/ml and 1.0 mg/ml) for which hF.IX expressionhad reached a plateau. FIG. 7 shows hF.IX serum concentrations at day 7post injection of naked hF.IX pDNA or hF.IX pDNA/poly-L-glutamic acidmixtures in the tibialis muscle of C57BL/6 mice and electroporation ofthe tissue. The amount of hF.IX pDNA administered per animal was either25 μg (0.5 mg/ml) or 50 micrograms (1.0 mg/ml) in 100 microliters (halfthis dose per leg). The concentration of the anionic polymer in theinjected solution varied as indicated on the graph. The poly-L-glutamicacids selected differed by their molecular weight, ranging from 0.5-1.5kDa (with a degree of polymerization (DP) of 9) to 15-50 kDa. Allpoly-L-glutamic acids tested were able to increase hF.IX expressionsubstantially, especially at 6.0 mg/ml, with only small differences inpotency between polymers. The highest hF.IX level obtained afterinjection in the tibialis muscle of C57BL/6 mice and electroporation ofthe tissue was 280 ng/ml, with a treatment consisting of DNA at 0.5mg/ml and the 2-15 kDa poly(L-glutamic acid) at 6.0 mg/ml. Incomparison, the naked DNA treatment only resulted in hF.IX levels around160 ng/ml.

Persistence of Expression from Plasmid DNA

To determine if hF.IX expression could persist in the plasma for anextended time in the absence of an immune response, plasmid formulatedwith PVP (5%) was tested in immune deficient SCID beige mice. FIG. 8shows hF.IX expression in plasma of immune deficient (SCID beige) mice.Mice were initially injected with plasmid (1 mg/ml) formulated with 5%PVP (25 microliters each tibialis muscle and 50 microliters in eachgastrocnemius muscle). Consistent with expression patterns in immunecompetent mice, hF.IX levels peaked 7 days after injection at ˜120 ng/ml(FIG. 8). Following a 35% drop in hF.IX levels by 14 days afterinjection, expression remained fairly stable to 90 days post injectionbut had fallen to ˜20% of peak values by day 125.

At day 153, the animals were re-injected with plasmid and electroporatedin the same muscles that were used in the first treatment. For thesecond injection at day 153 (indicated by the arrow), the animals wereseparated into two groups. One group was injected with plasmidformulated with 5% PVP (n=7) and the other group injected with plasmidformulated with 6 mg/ml poly-L-glutamate (n=8). The second injectionsutilized the same injection sites and plasmid dose that were used forthe first injections. In both groups of SCID mice, plasmidre-administration led to a significant rise in plasma hF.IX levels. Thegroup injected with plasmid formulated with poly-L-glutamate hadsignificantly higher expression than the group injected with PVP. Thisdifference in expression levels between the groups following the secondadministration was maintained throughout the duration of the experiment.The kinetics of hF.IX expression in both groups were similar to thatseen after the first administration in that there was a significant dropfrom peak expression (obtained ˜7 days after re-injection) within thefirst two weeks.

The graphs in the insert of FIG. 8 also show the effect of 6 mg/mlpoly-L-glutamate on hF.IX and hEPO expression in comparison to saline.For these experiments, the tibialis of mice were injected with plasmidcoding for hF.IX (50 micrograms) or for human erythropoietin (75micrograms) followed by electroporation. Plasma or serum samples werecollected 7 days after treatment for analysis. All values arerepresented as mean±SEM. A Students t-test was used to compare means andin FIG. 8, *=P≦0.05. Plasmids formulated with poly-L-glutamate (6 mg/ml)led to a 1.5 fold to 5.9 fold enhancement in expression compared toplasmid in saline with electroporation and was dependent on the insertedgene (FIG. 8, insert).

In the SCID mice at 10 months after the initial injection with PVPfollowed by reinjection with a poly-L-glutamate formulation, thetibialis and gastrocnemius muscles were harvested for hF.IXimmunostaining and muscle fiber typing. FIG. 9 shows immunohistology andfiber-type of hF.IX expressing myocytes in SCID mouse muscle.Representative sections of SCID mouse gastrocnemius muscle from tissuethat was harvested ˜300 days after the initial injection. FIG. 9A showshF.IX immunolocalization wherein positive myocytes are stained dark(original magnification 100×). FIG. 9B shows ATPase staining (pH 4.6) ofa serial section of panel A. Type I fibers (dark) and type II fibers(light) are distinguished (original magnification 100×). Arepresentative sample of complementary fibers are labeled in both panelsindicating both type I and type II fibers are expressing hF.IX. Both thetibialis and gastrocnemius muscles showed a broad distribution of fibersexpressing hF.IX. In the gastrocnemius, expression was found in bothtype I and type II fibers in roughly equal proportions although theabsolute number of stained type I fibers was much lower than type IIfibers (FIG. 9). In the mouse tibialis there were few if any type Ifibers and thus expression was observed primarily in type II fibers.Thus, long-term expression of hF.IX, achieved in immune compromised(SCID beige) mice, indicates that plasmids are stable andtranscriptionally active in muscle for a prolonged period of time.

Applicability to Large Animals

The applicability of the gene delivery procedure to large animals is anecessary prerequisite step for the development of a potentiallyclinically useful gene therapy. FIG. 10A depicts the results of plasmahF.IX levels in dogs following intramuscular injection of plasmidaugmented by electroporation. Six adult dogs (beagles 9-13 kg) wereinjected with ˜1.6 or ˜2.8 mg/kg of plasmid using a multiple siteprotocol and followed by electroporation with 6-needle array electrodes.The DNA was formulated with poly-L-glutamate (6 mg/ml) for thesestudies. The dogs were divided into two groups. In one group a totaldose of 18 mg was administered intramuscularly divided into 6 sites, onein each of the biceps femoris, semimembranosus and cranial tibialismuscles of both rear legs. In the second group, 36 mg of plasmid wasadministered intramuscularly into 12 sites, one each in the bicepsfemoris, semimembranosus, semitendinosus, vastus lateralis, cranialtibialis and long head of the triceps brachii muscles of the front andrear limbs. A total volume of 2.0 ml was administered to each site. Ateach site 2.0 ml of plasmid (1.5 mg/ml) formulated with 6.0 mg/mlpoly-L-glutamate was injected followed by electroporation with a6-needle array electrode. The 6 and 12 injection site groups had 18 mgand 36 mg of plasmid injected per animal, respectively. FIG. 10A showsthe results where plasma was collected and analyzed by ELISA. Values aremeans±SEM with n=3 for each group.

Mean values of the 12 and 6 injection site groups peak at 36.1 ng/ml(day 22) and 27.2 ng/ml (day 14), respectively (FIG. 10A). The valuesfor the two groups diverged at day 22 due to an unexpected increase inmean expression in the group of animals injected at 12 sites. However,the expression levels in this group at day 22 are not significantlyhigher than at day 14. Regardless of this anomaly, by day 28 expressionlevels of both groups were indistinguishable from background levels.

Immune Response to Expressed Protein

FIG. 10B shows a western blot of purified hF.IX using treated animalserum as the primary antibody. Lane A represents the molecular weightmarker; lane B represents the negative control (i.e., serum fromuntreated animals); lane C represents the positive control (i.e., canineserum spiked with rabbit anti-hF.IX antibodies); lane D represents theimmunoreaction to hF.IX by the serum from a female dog from the 6injection group (peak expression hF.IX 35.71 ng/ml); lane E representsthe immunoreaction to hF.IX by the serum from a male dog from the 12injection group (peak hF.IX expression 47.9 ng/ml). Thus, analysis byWestern blot indicated that plasma from the dogs contained material thatcross-reacted with purified hF.IX consistent with an immune response tothe human protein (FIG. 10B).

Furthermore, serum analysis also revealed a transient increase increatine kinase (CK) levels that peaked two days after treatment, andreturned to normal levels by 7 days after treatment indicating somemuscle trauma is associated with the gene delivery procedure usinginvasive 6-needle array electrodes. This response is clearly dosedependent with the animals administered the higher dose (12 injectionsites) having higher peak levels of CK on day 3 than did the animalsfrom the 6 injection sites group. A histological examination of thedifferent injected muscles revealed some muscle damage approximately 1month after treatment. In most instances, no histological changes werenoted or were restricted to small focal points, where there wereindications of myocyte loss and infiltrating monocytes. In rareinstances, the injection site was characterized by areas of necrotictissue and associated myocyte loss. This type of damage was alsoobserved in mice at earlier time points after treatment (2 weeks) whenthe caliper electrodes were used, but the muscles recovered to normalhistology over time (data not shown). There was no indication that aparticular muscle type was more susceptible to tissue damage thananother.

Expression is Dose Dependant

To establish that expression of hF.IX in canine muscle wasdose-dependent, biceps femoris and tibialis cranialis of the left andright hindlimbs of 11-week-old dogs were used for the gene deliveryprotocol. Formulated plasmid was injected into 4 sites in each dog (leftand right tibialis cranialis, left and right biceps femoris). Theplasmid concentration was 3.0 mg/ml. Injected volumes (at each site)were 0.12 ml, 0.36 ml, 0.60 ml and 1.2 ml for each group. Serum wascollected 7 days after treatment for analysis (peak levels). Tonormalize for variations in the animals' weight, absolute hF.IX levelsare represented (determined by estimating blood volume at 7% of the dogsweight). Values are means±SEM with n=3 for each group. Values aremeans±SEM per animal with n=4 for each group. Plasma hF.IX levelsincreased with increasing amounts of plasmid from 0.8 mg/kg up to 2.3mg/kg. At high doses of plasmid (5.3 mg/kg) mean expression levels werelower than obtained at the 2.3 mg/kg dose but the difference was notsignificant.

Using plasmid injected into skeletal muscle followed immediately withelectroporation, we have achieved therapeutically significant levels ofhF.IX expression in the plasma of mice and dogs.

Optimized hF.IX Sequence

The above experiments were performed with plasmid pFN0945 (SEQ. ID. NO.3 and FIG. 17), which has the natural human nucleic acid sequenceencoding for hF.IX. For gene therapy applications in human, pFN0945 mayalso be used, but a codon optimized sequence for hF.IX may be preferredwhen higher expression is desired due to higher translation of a codonoptimized mRNA. An example of a codon optimized sequence for hF.IX isplasmid pFN1645, which is disclosed as SEQ. ID. NO. 4 and shown in FIG.18.

Example IV Expression of Therapeutic Genes

The ability of poly-L-glutamate to increase the expression of anon-viral erythropoietin (“EPO”) gene was also undertaken. Usingquantitative polymerase chain reaction (qPCR) analysis, plasmidformulated in Poly-L-Glutamate resulted in at least a log increasedlevels of mEPO DNA compared with animals receiving a saline/DNAformulation.

EPO Expression Using Polymer Formulations

The mEPO coding sequence was inserted into the Valentis plasmid backbonecontaining a 107 bp 5′ UTR, a 117 bp synthetic intron, the human growthhormone polyadenylation signal, a PUC 12 origin of replication and akanamycin resistance gene as aforementioned. The mEPO gene was driven bythe CMV enhancer/promoter. The complete sequence of the resultingplasmid pEP1403 containing the mEPO gene is disclosed in the sequencelisting as SEQ. ID. NO. 2 and the plasmid map is shown in FIG. 19.Plasmids were grown in Escherichia coli DH5 and were purified using aproprietary method involving alkaline lysis and chromographic methods(Abruzzese, R. V., et al. (1999) Hum Gene Ther 10:1499-1507,incorporated herein by reference).

Animals received CMV-mEPO formulated either in 15-50 kDapoly-L-glutamate or in saline. Plasmid formulations were injectedintramuscularly in each leg, 25 microliters in each tibialis, 50microliters in each gastrocnemius followed by electroporation 2 minafter injection (375 V/cm (113 V/0.3 cm), 2 pulses, 25 msec pulselength. At defined time intervals, blood was collected by retro-orbitalmethods and hematocrit levels determined or the serum assayed for EPOlevels.

At indicated times, total muscle DNA was extracted and levels of werequantified by qPCR as follows: Plasmid DNA quantities in mouse muscleswere determined by conducting TaqMan real time quantitative PCR (AppliedBiosystems, Foster City, Calif.) on isolated DNA samples as previouslydescribed (Mahato, R. I. et al. Hum. Gene Ther. 9, 2083-2099 (1998)).The primers used in the PCR were a forward primer, which primes in the5′ untranslated region, and a reverse primer, which primes in the mouseEPO coding region. The probe sequence was located within the EPO gene.Purified CMV-mEPO plasmid DNA was used to generate a standard curve forthe PCR assay. As shown in FIG. 11, formulation in poly-L-glutamateresults in a several fold increase in the amount of plasmid DNA that canbe detected in tissues after electroporation.

For mEPO expression determination, 75 mg pEP1403 (SEQ. ID. NO. 2) in 150ml was delivered to C57BL/6 mice, 25 microliters per tibialis, 50microliters per gastrocnemius. Plasmid was formulated in saline or 6mg/mL poly-L-glutamate. FIGS. 12 and 13 depict mEPO expression and FIG.12 also depicts the hemotocrit level in mice following delivery of themouse EPO gene by electroporation using saline and sodiumpoly-L-glutamate formulations.

As shown in FIGS. 12 and 13, delivery in a poly-glutamate formulationresults in considerably higher levels of expressed protein than when theplasmid DNA is delivered in saline. Because a very small amount oferythropoietin is required to give a maximal increase in hematocrit, theinduced hematocrit levels shown on FIG. 12 do not differ between salineand polyglutamate formulations. However, because polyglutamate resultsin more efficient transfection, it is expected that lower amounts of DNAcan be administered using polyglutamate formulations.

Example V Expression of Therapeutic Genes Interferon Alpha ExpressionUsing Polymer Formulations

The hINFα 2b coding sequence was inserted into the Valentis plasmidbackbone containing a 107 bp 5′ UTR, a 117 bp synthetic intron, thehuman growth hormone polyadenylation signal, a PUC 12 origin ofreplication and a kanamycin resistance gene. The hINFα gene was drivenby the CMV enhancer/promoter. The complete sequence of the resultingplasmid pIF0921 containing the hINF-α gene is disclosed in the sequencelisting as SEQ. ID. NO. 1 and the plasmid map is shown in FIG. 20.Plasmids were grown in Escherichia coli DH5α and were purified using aproprietary method involving alkaline lysis and chromographic methods(Abruzzese, R. V., et al. (1999) Hum Gene Ther 10:1499-1507,incorporated herein by reference).

For expression analysis, 25 microliters plasmid formulations either inpoly-glutamate or in saline that had varying DNA concentrations (1.0mg/ml, 0.1 mg/ml and 0.01 mg/ml) were injected into each tibialis-bothlegs were electroporated with caliper electrodes at 375V/cm, 2 pulses,25 ms each pulse. For analysis, serum was collected via retro orbitalbleeds (days 4, 7, 14 and 30). A commercially available ELISA (Endogen)was used to determine INF-α levels. As shown in FIGS. 14A and B, asignificant enhancement of hINF-α expression in CD-1 mice was obtainedusing plasmid formulated with 6 mg/ml poly-L-glutamate at both 5 and 50microgram DNA doses.

Example VI Nuclease Protection of Plasmid DNA Formulated inPoly-L-Glutamate

Experiments were undertaken to determine the ability of poly-L-glutamateand Pluronic F68 to protect plasmid DNA from nuclease digestion. DNase Iwas obtained from Gibco/BRL (#18068-015). The sodium salt ofpoly-L-glutamic acid, 2-15 kDa was obtained from Sigma. Pluronic F68 wasobtained from Spectrum. Polymer/DNA2× stock solutions were prepared(Pluronic F68=200 micrograms/ml plasmid DNA in 10% F68;Poly-L-glutamate=200 micrograms/ml plasmid DNA in 12 mg/ml sodiumpoly-L-glutamate). DNase dilutions from 1:10 to 1:10,000 were preparedin 1× DNase buffer. The final reaction mixtures included 25 microlitersof the formulation, 15 microliters of water, 5 microliters of 10× DNasebuffer and 5 microliters of Dnase that were added in the order listed.The reaction mixtures were incubated for 15 minutes at 37° C. andterminated by addition of EDTA prior to gel electrophoresis.

The results of the DNase protection assay are shown in FIG. 15. Panel Arepresents a DNA in saline formulation; Panel B represents DNAformulated in 5% Pluronic F68; Panel C represents DNA formulated in 6mg/ml poly-L-glutamate. Lane A represents the negative control (i.e.,plasmid DNA without Dnase); lane B represents the positive control(i.e., plasmid DNA and DNase mixed 1:1); lanes C-G represents theexperimental conditions wherein DNA formulated with either saline (PanelA), F68 (Panel B), or poly-glutamate (Panel C) were mixed with DNasediluted 1:1 (lane C); 1:10 (lane D);1:100 (lane E); 1:1,000 (lane F);and 1:10,000 (lane G). In saline, DNase at 1:100 is able to abolish thelower band of supercoiled plasmid in addition to degradation of the DNAresulting in a smear of different molecular weights on the gel. Incontrast, both poly-L-glutamate and Pluronic F68 were able to conferprotection from DNase degradation at 1:100 dilution.

Example VII Long-Term Biological Stability of DNA formulated inPoly-L-Glutamate

Experiments were also undertaken to evaluate the stability of liquidpoly-L-Glutamate (15-50 kDa)/DNA formulations.

Animals: 108 CD-1 mice (29-31g) were obtained from Charles Rivers Labs.The animals were housed in microisolators (10 mice per isolator) in theLaboratory Animal Resource (LAR) vivarium and maintained at 12/12 hday/night cycle, room temperature 72° F. (23° C.), and humidity 40%.Food (Purina rodent chow) and water was provided ad libitum. Combinationanesthesia consisting of a mixture of Ketamine (74.0 mg/ml), Xylazine(3.7 mg/ml), and Acepromazine (0.73 mg/ml) was administered IP at adosage of 1.8-2.0 ml/kg.

Treatment Groups and Routes of Administration: The animals were randomlydivided into treatment groups with 6 (tibialis) or 5 (gastrocnemius)mice/group. For the tibialis groups, 25 microliters of the formulationsdescribed below were injected in each tibialis muscle, i.e. 50microliters in total volume per mouse. For the gastrocnemius groups, 50microliters of the formulations described below were injected in eachgastrocnemius muscle, i.e. 100 microliters in total volume per mouse.

Formulations: Formulations were prepared in 150 mM NaCl, 5 mM Tris-HCl,pH 7.5. SEAP encoding plasmid pAP1166.157 at 1 mg/ml was used. Plasmidand poly-L-Glutamate (15-50 kDa) were formulated as follows.

pDNA conc. Formulation (mg/ml) salt Poly-L-Glu Buffer A 1.0 150 mM 6.0mg/ml 5 mM Tris/pH 7.5 B 0 150 mM 6.0 mg/ml 5 mM Tris/pH 7.5

For the liquid formulations, A (0.5 ml) and B (1.5 ml) of the samestorage conditions were mixed (or rehydrated with water and mixed forthe lyophilized samples) right before use for in-vivo testing (in thegastrocnemius and tibialis muscles of CD-1 mice) and QC analysis. Thefinal DNA concentration of the mixture was 0.25 mg/ml. Each An/Bn couplewas tested at day 8, 21, 60 and 105. As a control, a fresh sample of 0.5ml of A and 1.5 ml of B was tested at every time point. As a fresh nakedDNA control, a sample of 0.5 ml of A (A not including poly-L-Glutamate)and 1.5 ml of B (B not including poly-L-Glutamate) was tested at everytime point.

The lyophilization/storage conditions for which results are shown inFIG. 16 were the following:

Group Physical storage condition Temperature A Lyophilization (storageN.A. for the  +4° C. sample tested right after completion of thelyophilization cycle) B Liquid −20° C. C Liquid  +4° C. D Liquid +25° C.E Liquid +37° C. F Liquid +50° C. G Liquid/storage with afreeze/thaw/freeze −20° C. cycle at day 2, 4 (and 10, 17, 24, 31, 38,45, 52 and 59 if applicable) H Fresh DNA/pGlu I Fresh naked DNA

FIG. 16 depicts the results of the final 105 day time point andindicates the biological activity of the DNA under different storageconditions. As indicated on FIG. 16, plasmid DNA at 1 mg/ml formulatedin poly-L-glutamate at 6 mg/ml is stable for over three months in liquidsolution at room temperature. Poly-L-glutamate also protected the DNAagainst degradation during freeze thawing and lyophilization.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art which are encompassed within the spirit ofthe invention are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationsthat is not specifically disclosed herein. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and there is no intention that in the use of such terms andexpressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

Those references not previously incorporated herein by reference,including both patent and non-patent references, are expresslyincorporated herein by reference for all purposes. Other embodiments arewithin the following claims.

1. A method of delivering a nucleic acid encoding a therapeutic geneproduct to a cell comprising: introducing the nucleic acid encoding thetherapeutic gene product to a cell in a formulation comprising thenucleic acid formulated with an anionic polymer selected from the groupconsisting of: poly glutamic acid, poly aspartic acid, poly galacturonicacid, poly vinyl sulfate, and co-polymers of glutamic and aspartic acid,and salts thereof, wherein the anionic polymer is non-encapsulating andthe formulation does not include a cationic polymer.
 2. The method ofclaim 1, wherein the anionic polymer is selected from the groupconsisting of poly glutamic acid and poly aspartic acid, and saltsthereof.
 3. The method of claim 1, wherein the anionic polymer ischaracterized by a molecular weight in the range from 2,000 to 100,000Daltons.
 4. The method of claim 3, wherein the anionic polymer ischaracterized by a molecular weight in the range from about 15,000 toabout 50,000 Daltons.
 5. The method of claim 3, wherein the anionicpolymer is characterized by a molecular weight in the range from about2,000 to about 15,000 Daltons.
 6. The method of claim 3, wherein theanionic polymer is characterized by a molecular weight in the range fromabout 50,000 to about 100,000 Daltons.
 7. The method of claim 1, whereinthe anionic polymer or salt thereof is formulated with the nucleic acidencoding the therapeutic gene product at a polymer concentration rangingfrom 1 to 12 mg/ml.
 8. The method of claim 7, wherein the anionicpolymer or salt thereof is formulated with the nucleic acid encoding thetherapeutic gene product at a polymer concentration of about 2 to about6 mg/ml.
 9. The method of claim 1, wherein the formulation is isotonic.10. The method of claim 1, wherein the formulated nucleic acid isintroduced to the cell in a tissue in vivo.
 11. The method of claim 10,wherein the tissue is a muscle tissue.
 12. The method of claim 1,further comprising a step of storing the formulated nucleic acid byliquid storage, lyophilization and/or freezing.
 13. The method of claim1, wherein the therapeutic gene product is selected from the groupconsisting of: mRNA templates for translation, ribozymes, antisense RNA,proteins, glycoproteins, lipoproteins, phosphoproteins and polypeptides.14. The method of claim 13, wherein the therapeutic gene product is aprotein selected from the group consisting of growth hormones, growthfactors, cytokines, clotting factors, antigens, and antigenic factors.15. The method of claim 14, wherein the clotting factor is a Factor IX.16. The method of claim 14, wherein the growth factor is anerythropoietin.
 17. The method of claim 14, wherein the cytokine is aninterferon.
 18. The method of claim 1, wherein the poly glutamic acid isa salt of poly-L-glutamic acid and is present in the formulation at aconcentration of 1 to 12 mg/ml.
 19. The method of claim 18, wherein thesalt of poly-L-glutamic acid is a sodium salt and is present in theformulation at a concentration of about 6 mg/ml.
 20. The method of claim10, wherein the formulated nucleic acid is introduced into the tissue invivo by injection.
 21. The method of claim 10, further comprising a stepof electroporating the tissue.
 22. The method of claim 21, wherein theelectroporating is performed through the use of a device configured andarranged to cause pulse voltage delivery of the formulated nucleic acid.23. The method of claim 22, wherein the pulse voltage delivery isperformed at a pulse strength in a range of about 200V/cm to about 1,800V/cm.
 24. The method of claim 23, wherein the pulse voltage delivery isperformed at a pulse strength selected from the group consisting ofabout: 200V/cm, 375V/cm, and 1,800 V/cm.
 25. The method of claim 22,wherein the pulse voltage delivery is performed at a pulse length in therange of about 100 microsec to about 25 millisec.
 26. The method ofclaim 25, wherein the pulse voltage delivery is performed at a pulselength selected from the group consisting of about: 100 microsec 25millisec, and 60 millisec.
 27. The method of claim 22, wherein the pulsevoltage delivery is performed using a plurality of pulses.
 28. Themethod of claim 27, wherein the plurality of pulses ranges from 2 toabout 6 pulses.
 29. The method of claim 22, wherein the pulse voltagedelivery is performed using a square wave pulse.
 30. The method of claim22, wherein the method induces an immune response.
 31. A method fordelivering a therapeutic gene product to a mammalian tissue, comprisingthe steps of: providing a non-condensing, non-encapsulating formulationcomprising a nucleic acid encoding the therapeutic gene product and ananionic polymer selected from the group consisting of: poly glutamicacid, poly aspartic acid, poly galacturonic acid, poly vinyl sulfate,and co-polymers of glutamic and aspartic acid, and salts thereof,introducing the formulation to the mammalian tissue in vivo; andelectroporating the tissue, whereby the nucleic acid is introduced intocells in the tissue and the therapeutic gene product is expressed. 32.The method of claim 31, wherein the electroporating is performed throughthe use of a device configured and arranged to cause pulse voltagedelivery of the formulated nucleic acid.
 33. The method of claim 32,wherein the pulse voltage delivery is performed at a pulse strength in arange of about 200V/cm to about 1,800 V/cm.
 34. The method of claim 33,wherein the pulse voltage delivery is performed at a pulse strengthselected from the group consisting of about: 200V/cm, 375V/cm, and 1,800V/cm.
 35. The method of claim 32, wherein the pulse voltage delivery isperformed at a pulse length in the range of about 100 microsec to about25 millisec.
 36. The method of claim 35, wherein the pulse voltagedelivery is performed at a pulse length selected from the groupconsisting of about: 100 microsec 25 millisec, and 60 millisec.
 37. Themethod of claim 32, wherein the pulse voltage delivery is performedusing a plurality of pulses.
 38. The method of claim 37, wherein theplurality of pulses ranges from 2 to about 6 pulses.
 39. The method ofclaim 32, wherein the pulse voltage delivery is performed using a squarewave pulse.
 40. The method of claim 31, wherein the tissue is a muscletissue.
 41. The method of claim 31, wherein the therapeutic gene productis selected from the group consisting of: mRNA templates fortranslation, ribozymes, antisense RNA, proteins, glycoproteins,lipoproteins, phosphoproteins and polypeptides.
 42. The method of claim41, wherein the protein is selected from the group consisting of growthhormones, growth factors, cytokines, clotting factors, antigens, andantigenic factors.
 43. The method of claim 42, wherein the clottingfactor is a Factor IX.
 44. The method of claim 42, wherein the growthfactor is an erythropoietin.
 45. The method of claim 42, wherein thecytokine is an interferon.
 46. The method of claim 31, wherein the polyglutamic acid is a salt of poly-L-glutamic acid and is present in theformulation at a concentration of 1 to 12 mg/ml.
 47. The method of claim46, wherein the salt of poly-L-glutamic acid is a sodium salt and ispresent in the formulation at a concentration of about 6 mg/ml.